During embryonic development, the sexually indifferent gonad chooses between adopting an ovarian or testicular fate. In XY individuals, the testicular fate is initiated by a gene on the Y chromosome, Sry, that triggers a cascade of events leading to development of the testis. Although no single gene that triggers the ovarian pathway has been identified in XX individuals, several genes have been identified that are required not only for development of ovarian structures, but also for inhibiting the male pathway. The earliest known gene in the ovarian pathway is Wnt4. Mice deficient in Wnt4 show partial female-to-male sex reversal, and both loss and gain-of-function mutations of human WNT4 cause abnormal sexual development. The long- term goals of this proposal will be to elucidate the signaling pathways that drive the sexually indifferent gonad towards an ovarian fate. Specifically, we aim to understand three aspects of Wnt4 signaling. Wnt signals are transmitted through a family of frizzled receptors. In the first aim we will identify the frizzled receptor mediating the Wnt4 signal by determining the expression of these various receptors in the female gonad and then testing for a direct interaction with the WNT4 ligand. Recent data suggests that a canonical Wnt signaling mechanism involving Beta-catenin is not active in the XX gonad. Specific aim 2 will investigate the role of Wnt4 in activating various non-canonical signaling pathways. Using an established gonad culture system, we will test whether a Wnt-mediated calcium signal functions in the XX gonad. The activation of a non-canonical, dishevelled-mediated pathway will also be tested by looking for interactions between dishevelled and other downstream components of the pathway. XX gonads lacking Wnt4 will be used to confirm that these signals are dependent upon WNT4 signaling in vivo. In specific aim 3 we will address the inhibitory role of Wnt4 on the male pathway. Previous experiments suggest that Wnt4 antagonizes SOX9 stabilization by blocking the co-localization of FGFR2 in the nucleus. To test this hypothesis, we will generate a transgenic mouse that expresses Fgfr2 from the Sox9 promoter. This model will test the antagonistic action of WNT4 against maintenance of SOX9 in the presence or absence of FGFR2. These experiments will extend our understanding of the role Wnt4 plays in promoting ovarian development while simultaneously antagonizing testis differentiation. Not only will this work illuminate critical mechanisms of organogenesis, but it will also reveal molecular explanations for perturbations in sexual development in humans. [unreadable] [unreadable] [unreadable]